The invention relates to thermocyclers for an automatic performance of polymerase chain reaction (PCR), particularly to rapid thermocyclers. More specifically, it relates to rapid heat block thermocyclers for parallel processing of multiple small-volume samples. The present invention is especially useful for rapid, high-throughput, inexpensive and convenient PCR-based DNA-diagnostic assays.
Since it""s first published account in 1985 polymerase chain reaction has been transformed into myriad array of methods and diagnostic assays. Temperature cycling of samples is the central moment in PCR. In recent years various rapid thermocyclers have been developed to address the slow processing speed and high sample volumes of conventional heat block thermocyclers. These rapid thermocyclers can be divided into two broad classes:
1. Capillary thermocyclers hold the samples within a glass capillary and supply heat convectively or conductively to the exterior of the capillary. For the description see Wittwer, C. T., et al., Anal.Biochem. 186: p328-331 (1990); Friedman, N. A., Meldrum, D. R. Anal. Chem., 70: 2997-3002 (1998) and U.S. Pat. No. 5,455,175.
2. Microfabricated thermocyclers are thermocyclers constructed of microfabricated components; these are generally etched structures in glass or silicon with heat supplied by integral resistive heating and rejected passively (or actively) to ambient by the structure. However, other schemes of thermocycling, as continuous flow thermocycling of samples are also used. For the description see Northrup, M. A., et al., Transducers 1993: 924-926 (1993); Taylor, T. B., et al, Nucleic Acid Res., 25: pp 3164-3168 (1997); Kopp, M. U. et al., Science, 280: 1046-1048 (1998); U.S. Pat. No. 5,674,742; U.S. Pat. No. 5,716,842.
Both classes of rapid thermocyclers employ the increased surface-to-volume ratio of the reactors to increase the rate of-heat transfer to small samples (1-20 xcexcl). Total DNA amplification time is reduced to 10-30 minutes. Conventional heat block thermocyclers usually take 1-3 hours to complete temperature cycling of 20-100 xcexcl samples. However, with these benefits also several disadvantages appear. Increased surface area between reagents and reactors causes a loss of enzyme activity. Furthermore, DNA can also be irreversibly adsorbed onto silica surface of the reactors, especially in the presence of magnesium ions and detergents that are the standard components of a PCR mixture. Therefore, PCR in glass-silicon reactors requires the addition of carrier protein (e.g. bovine serum albumin) and a rigorous optimization of the composition of the reaction mixture.
Another disadvantage of these reactors is the very complicated way of loading and recovering the samples. In addition, standard pipetting equipment is usually not compatible with such reactors. These inconvenient and cumbersome procedures are also time-consuming and labor-sensitive, thus limiting the throughput of the thermocyclers. Finally, although the reagents costs drop with a volume reduction to 1-10 xcexcl, the final costs are relatively high due to a high cost of capillary and, especially, microfabricated reactors.
Therefore, it is surprising that only little research has been conducted to improve the basic performance in sample size and speed of the widely used, conventional heat block thermocycling of samples contained in plastic tubes or multiwell plates. One known improvement of heat block temperature cycling of samples contained in plastic tubes has been described by Half et al. (Biotechniques, 10, 106-112, [1991] and U.S. Pat. No. 5,475,610). They describe a special PCR reaction-compatible one-piece plastic, i.e. polypropylene, microcentrifuge tube, i.e. a thin-walled PCR tube. The tube has a cylindrically shaped upper wall section, a relatively thin (i.e. approximately 0.3 mm) conically- shaped lower wall section and a dome-shaped bottom. The samples as small as 20 xcexcl are placed into the tubes, the tubes are closed by deformable, gas-tight caps and positioned into similarly shaped conical wells machined in the body of the heat block. The heated cover compresses each cap and forces each tube down firmly into its own well. The heated platen (i.e. heated lid) serves several goals by supplying the appropriate pressure to the caps of the tubes: it maintains the conically shaped walls in close thermal contact with the body of the block; it prevents the opening of the caps by increased air pressure arising in the tubes at elevated temperatures. In addition, it maintains the parts of the tubes that project above the top surface of the block at 95xc2x0-100xc2x0 C. in order to prevent water condensation and sample loss in the course of thermocycling. This made it possible to exclude the placing of mineral oil or glycerol into the wells of the block in order to improve the heat transfer to the tubes and the overlaying of the samples by mineral oil that prevented evaporation but also served as added thermal mass. In addition, the PCR tubes can be put in a two-piece holder (U.S. Pat. No. 5,710,381) of an 8xc3x9712, 96-well microplate format, which can be used to support the high sample throughput needs with any number between 1 and 96 individual reaction tubes. When compared to conventional microcentrifuge tubes the use of thin-walled 0.2-ml PCR tubes made it possible to reduce the reaction time from 6-10 hours to 2-4 hours or less. At the same time it was also shown in DE 4022792 that the use of thin-walled polycarbonate microplates allows to reduce the reaction time to less than 4 hours. A recent improvement concerning the ramping rate (i.e. 3-4xc2x0 C./second) of commercial thermoelectric (Peltier effect) heat block thermocyclers did not influence considerably the total reaction time. Moreover, it was concluded that a further increase in ramping rates will not be of a practical benefit due to the limited rate of heat transfer to the samples contained in thin-walled PCR tubes (see WO 98/43740).
The present invention bears some similarity to conventional heat block thermoelectric thermocyclers for performing PCR in plastic microplates (for example, see WO 98/43740 and DE 4022792). However, in contrast to conventional heat block thermocylers, it provides the means for performing PCR, i.e. 30 cycles, in 1-20 xcexcl samples in 10-30 minutes. More specifically, it provides a rapid heat block thermocycler for convenient, high-throughput and inexpensive, oil-free temperature cycling of multiple small-volume samples.
Accordingly, the invention concerns a heat block thermocycler for subjecting a plurality of samples to rapid thermal cycling, the heat block thermocycler including:
a unit for holding a plurality of samples having
an ultrathin-walled multiwell plate having an array of conically shaped wells and a low thermal mass sample block having an array of similarly shaped wells, wherein the height of the wells of the said multiwell plate is not more than the height of the wells of the said sample block,
a unit for heating and cooling the sample block comprising at least one thermoelectric module, and
a device for sealing the plurality of samples comprising a high-pressure heated lid.